Microbiology: Prokaryotic Growth

What is the minimum number of bacteria cells required to form a colony?

A single viable bacterial cell

Each distinct, visible colony on an agar plate is assumed to originate from one mother cell multiplying through binary fission.

Describe the process of binary fission

1. Cell gets longer and DNA replicates

2. DNA is moved into each daughter cell and cross wall forms

3. Cell divides into 2 cells’

4. Cells separate

5. Form genetically identical daughter cells

Why do we say that binary fission is natural cloning?

It results in the production of genetically identical offspring from a single parent cell without any human intervention or genetic engineering

Why do microbes grow as mixed communities in nature?

Individual species rarely possess all the metabolic tools needed to survive harsh, changing, or nutrient-limited environments on their own

What are biofilms and why are they clinically significant?

a structured, cooperative community of microorganisms (such as bacteria or fungi) adhere to a surface and are encased in a self-produced slimy matrix

Biofilms are antibiotic resistant due to their thick matrix and can stick around for long periods in the body

Define Generation Time

Time required for a bacterial population to double in number

Define Pure Culture

a laboratory culture that contains only a single species or strain of microorganism

Define Mixed Culture

a laboratory sample or growth medium that contains two or more different species or strains of microorganisms growing together at the same time

Describe two methods for isolating pure cultures from a mixed culture.

Streak Plate Method: used to separate mixed bacteria into distinct categories

Spread Plate Method: used to count exactly how many living bacteria are in a liquid sample

How does the streak plate method work?

1. Sterilize loop

2. Dip loop into culture'

3. Streak first area of the agar

4. Sterilize loop

5. Streak second area of the agar

6. Sterilize loop

7. Streak final area of agar

8. Isolated colonies develop after incubation

How does the Spread Plate Method work?

1. Drop: Place a tiny drop of liquid bacteria right into the center of the plate.

2. Flame: Dip a small glass rod (shaped like a mini hockey stick) into alcohol and pass it through a flame to sterilize it.

3. Spread: Press the sterile rod onto the plate and spin the dish to sweep the liquid evenly across the entire surface.

4. Grow: Put the lid on and place the plate in a warm incubator overnight so individual dots (colonies) can grow

What is the difference between a continuous culture and a batch culture?

Continuous culture: Open system

• nutrients are replaced and waste products are removed

Batch culture: Closed System

• Nutrients not replaced and waste products are not removed

• Predictable growth pattern

When is a continuous culture used?

In large industrial settings for big products

What are different phases of a bacterial growth curve and what are the characteristics of each phase?

Lag phase: make macromolecules needed for growth in new media

Log (exponential) phase: optimal generation time, primary and secondary metabolites

Stationary phase: number of new cells is equal to dying cells

Death phase: cells dying at constant exponential rate

Prolonged decline phase: gradual decrease in viable cells, fitter cells emerge

List the environmental factors that influence microbial growth.

Temperature

O2 availability

pH

Water availability

What are the different categories of bacteria according to temperature?

Psychrophile

Psychotroph

Mesophile

Thermophile

Hyperthermophile

What is the growth range of each category?

Psychrophile: -5 to 15 degrees C

Psychotroph: 15 to 30 degrees C; grows well at refridgeration temps

Mesophile: 25 to 45 degrees C

Thermophile: 45 to 70 degrees C

Hyperthermophile: 70 degrees C or higher

Which category of bacteria includes the majority of human pathogens?

Mesophiles

What is the difference between an organism’s optimal growth temperature and its temperature growth range?

Optimal Growth Temperature: The specific point where cellular enzymes and proteins function at peak efficiency and reproduction is fastest.

Temperature growth range: The full spectrum bounded by the minimum growth temperature and the maximum growth temperature.

Why can’t bacteria grow at temperatures outside their range?

Extreme heat and cold break down the internal machinery they need to survive and reproduce

Growth outside the optimum but within this range is much slower or stressed.

How are bacteria classified according to oxygen requirements?

Aerobes and Anaerobes

What group of bacteria can use oxygen to produce energy? What group cannot?

Aerobes use oxygen

Anaerobes cannot use oxygen

What are the different categories of aerobic bacteria and what are their characteristics?

Obligate Aerobes: grows only when O2 is available, needs O2 for respiration, produces superoxide dismutase and catalase

Microaerophiles: grows only if small amounts of O2 are available, needs O2 for respiration, produces some superoxide dismutase and catalase

Facultative anaerobes: grows best with O2 but can grow without it, if O2 is available it uses it for respiration, produces superoxide dismutase and catalase

Capnophiles: CO2 incubator

What are the categories of Anaerobes and their characteristics?

Obligate Anaerobes: Cannot grow with O2 present, no O2 for respiration, no superoxide dismutase or catalase

Aerotolerant Anaerobes: Grows equally well with/without O2, no O2 for respiration, produces superoxide dismutase no catalase

Why aren’t aerobes killed in the presence of oxygen while obligate anaerobes are?

Aerobes can detoxify

When they detoxify they can produce highly destructive molecules such as superoxide and peroxide

They have lots of SOD and Catalase

What enzymes help bacteria detoxify oxygen and what reactions do these enzymes catalyze?

Superoxide Dismutase (SOD)

• It takes the most dangerous, aggressive free radicals (superoxides) and turns them into a milder chemical (hydrogen peroxide).

  • Dangerous Radical —> Hydrogen Peroxide + Safe Oxygen

Catalase

• Hydrogen peroxide is still toxic. Catalase steps in to instantly smash it down into completely harmless water and oxygen gas.

  • Hydrogen Peroxide —> Water + Safe Oxygen Gas

Peroxidase

• This does the same job as catalase but uses a slightly different chemical trick that converts it to water without making any oxygen gas bubbles.

  • Hydrogen Peroxide + Helper Molecule —> Water

Why can’t bacteria grow outside their normal pH range?

extreme acidity or alkalinity completely destroys their internal cellular machinery

Define an acidophile

An organism that grows optimally in acidic environments, typically at a pH value below 5.5.

Define an Alkalophile

An organism that grows optimally in alkaline/basic environments, typically at a pH value above 8.5.

What is the difference between an osmotolerant bacterium and one that is a halophile?

Osmotolerant ("Solute-Tolerator")

• It prefers normal conditions, but it can tolerate high amounts of salt or sugar if it has to. also no plasmolysis

• It uses internal chemical tricks to keep from drying out when things get sticky or salty, but it grows best in plain water.

Halophile ("Salt-Lover")

• It must have lots of salt to survive.

• Its entire body is built specifically for salt. If you put a halophile into regular, fresh water, it will actually pop and die.

Why is the growth of non-osmotolerant bacteria inhibited when placed in hypertonic solutions?

The growth of non-osmotolerant bacteria is inhibited in a hypertonic solution because they rapidly lose internal water and dehydrate through osmosis

What six elements are essential for all living organisms?

CHNOPS: >95% of microbial make up; Non fastidious

• Carbon

• Oxygen

• Hydrogen

• Nitrogen

• Sulfur

• Phosphorus

What biological molecules are these essential elements used to make?

C,O,H: amino acids, lipids, nucleic acids, sugars

N: amino acids and nucleic acids

S: some amino acids

P: nucleic acids, membrane lipids, ATP

What is a growth factor?

Compounds required for growth

Give examples of growth factors

Purines, Pyrimidines, Vitamins, Amino acids

What term is used to describe bacteria that require the addition of growth factors in order to grow?

Fastidious Bacteria

What is the energy source of phototrophs? Of chemotrophs? Of lithotrophs?

Phototrophs: Sunlight

Chemotrophs: chemical compounds

Lithotrophs: Inorganic chemical compounds

What is the carbon source of autotrophs? Of heterotrophs?

Autotrophs: uses carbon dioxide as carbon source

Heterotrophs: uses organic compounds as carbon source

Be able to list the carbon and energy sources for the following categories of

bacteria: photoautotrophs, photoheterotrophs, chemolithoautotrophs,

chemoorganoheterotrophs.

Photoautotrophs: Sunlight and CO2

Photoheterotrophs: Sunlight and organic compounds

Chemolithoautotrophs: Inorganic chemicals and CO2

Chemoorganoheterotrophs: organic chemicals and organic compounds

Be able to describe the following types of growth media: complex, enriched,

chemically defined, selective, differential, selective and differential

Complex: exact comp. not known

Chemically defined: exact comp. known

Selective: inhibits growth of some microbes and selects for the growth of others

Differential: be able to visually differentiate bacteria by growth characteristics

Enriched: contains additional nutrients to support growth of fastidious bacteria

Selective-Differential: plate kills off unwanted background microbes so target group can grow. Among bacteria that actually grow, the plate changes colors based on each bacterium's specific metabolic reactions.

Why is blood agar considered an enriched, differential medium?

it provides extra nutrients to help picky bacteria grow, and it visually separates bacteria based on how they destroy red blood cells

What methods measure both viable and non-viable cells?

Direct Cell Counts

• Counting Chambers: known vol.

• Coulter counters: changes in resistance

• Flow cytometry: scattering of light, dyes to count specific cells

What methods measure just viable cells?

Viable cell counts

• plate counts

• membrane filtration

For what types of samples is membrane filtration used?

Used for samples with low numbers of bacteria

sample passed through a filter

• concentrates bacteria in sample

Filter placed on media that will support growth

• incubate, count colonies

What method detect changes in cell biomass?

Turbidity

What is turbidity and how is it measured?

Cell density in a liquid culture

Measured with a spectrophotometer that measured absorbance

What is the relationship between turbidity and cell density?

Turbidity and cell density share a direct, proportional relationship

as concentration of bacteria in a liquid increases, the cloudiness or haziness of the liquid rises right along with it.